CN104540527A - Inhibitors of the MIR-15 family of micro-RNAs - Google Patents
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Abstract
The invention provides chemically modified oligonucleotides capable of inhibiting the expression (e.g., abundance) of miR-15 family miRNAs, including miR-15a, miR-15b, miR-16, miR-195, miR-424, and miR-497. The invention provides in some embodiments, oligonucleotides capable of inhibiting, in a specific fashion, the expression or abundance of each of miR-15a, miR-15b, miR-16, miR-195, miR-424, and miR-497. The invention further provides pharmaceutical compositions comprising the oligonucleotides, and methods of treating patients having conditions or disorders relating to or involving a miR-15 family miRNA, such as a cardiovascular condition. In various embodiments, the oligonucleotides provide advantages in one or more of potency, efficiency of delivery, target specificity, toxicity, and/or stability.
Description
Related application
The application advocates the U.S. Provisional Application the 61/662nd that on June 21st, 2012 submits to, the U.S. Provisional Application the 61/780th that No. 772 and on March 13rd, 2013 submit to, and the priority of No. 352 and rights and interests, it is quoted in full separately and is incorporated herein.
Electronics submits the description of text to
Electronics of the present invention submits to the content of text to be incorporated to herein by quoting in full: the computer-reader form copy of sequence table (filename: MIRG-037_02US_SeqList_ST25.txt, record date on June 19th, 2013, file size 10 kilobytes.)
Invention field
The invention relates to the chemical motif (motif) of Microrna (miRNA or miR) inhibitor.More particularly, the invention relates to the oligonucleotide of chemical modification, the one or more miR-15 family member of its targeting, and there is when giving patient the benefit of effect, delivery efficiency, targeting specific, stability and/or toxicity.
Background of invention
Microrna s (miR) has involved in multiple bioprocess, comprise regulate and safeguard cardiac function (see people such as E.Van Rooij., J.Clin.Invest.117 (9): 2369-2376 (2007); K.Chien, Nature 447:389-390 (2007)).Therefore, miR represents for the relatively new therapeutic target thing of a class of the situations such as such as cardiac hypertrophy (cardiachypertrophy), myocardial infarction (myocardial infarction), heart failure (heart failure), blood vessel injury (vascular damage) and pathological heart fibrosis (pathologic cardiac fibrosis).MiR is the tiny RNA of the long nonprotein coding of about 18 to about 25 nucleotide, and when with during its said target mrna complete complementary by promoting that said target mrna degrade, or pass through suppression when their sequence contains mispairing and translate, serve as the inhibitor of said target mrna.Its mechanism relates in the silencing complex (RISC) ripe miRNA chain being incorporated into RNA induction, is wherein combined with target RNA by base pair complementarity.
Can by antisense polynucleotides or polynucleotide (miRNA simulant) the therapeutic targeting miRNA function by simulation miRNA function.But, with the reagent targeting process miRNA based on oligonucleotide, there is some challenges, comprise RNA binding affinity and specificity, the efficiency of cellular uptake, nuclease resistant.Such as, when polynucleotide import in intact cell, they are degraded by nuclease attack, to cause active loss.Although prepared polynucleotide analog to attempt to avoid by its degraded, such as replace (the people such as B.Sproat by 2 ', Nucleic Acids Research 17:3373-3386, (1989)), this modification often affects the effect of polynucleotide for its predetermined biological agent (intended biologicalaction).In every case, the effect of this kind of minimizing is attributable to modified polynucleotide and can not forms stable Double helix and/or the loss with cellular machineries interphase interaction with target RNA.Other are modified and comprise lock nucleic acid (locked nucleic acid), and it has the potential improving RNA binding affinity.(see, the people such as R.Veedu., RNA Biology 6 (3): 321-323 (2009)).
There is for the oligonucleotide chemistry pattern of miRNA inhibitor or motif the potential improving the sending of inhibitor, stability, effect, specificity and/or toxicity form, and these are effectively needed for targeting miRNA function in treatment situation.
Summary of the invention
The invention provides the oligonucleotide of chemical modification, it can reduce or suppress to comprise miR-15a, miR-15b, miR-16, miR-195, miR-424, and miR-497 is in the activity of interior one or more miR-15 family members.The present invention further provides the compositions comprising oligonucleotide, and treatment suffer from one or more miR-15 family member about or relate to the method for one or more situation of miR-15 family member or the experimenter of disease.In each embodiment, disclosed oligonucleotide effect, delivery efficiency, target-specific, toxicity and/or stability one or more in benefit is provided.
On the one hand, the invention provides a kind of chemical modification oligonucleotide that can reduce or suppress the activity of one or more miR-15 family member.The activity of oligonucleotide or effect can in vitro and/or in vivoassay.Such as, at about 50nM or lower, about 40nM or lower in other embodiments, about 20nM or lower, or during the concentration of about 10nM or lower, the activity (as such as analyzed or dual luciferase analyses with two step real-time fluorescence quantitative PCRs) of oligonucleotide can significantly suppress (such as, about 50% suppress) one or more miR-15 family member.Or, or in addition, the activity of oligonucleotide can at the mice be applicable to or rat model, or measure in non-human primate model, wherein at about 50mg/kg or lower, all 25mg/kg according to appointment or lower, about 10mg/kg or lower, or under the dosage of about 5mg/kg or lower, observe suppression (such as, at least about 50%) to one or more miR-15 family member.This document describes the exemplary indicia thing for measuring target thing removal of inhibit function (de-repression).In these embodiments, oligonucleotide can through subcutaneous or intravenous (as described in this article) give with, and can with preparation in aqueous formulation (such as, saline).
Oligonucleotide comprises nucleotide sequence 5 '-GTGCTGCT-3 ', and complementary in fact with the nucleotide sequence of one or more miR-15 family member.Oligonucleotide is further containing at least one lock nucleotide.In one embodiment, oligonucleotide contains at least 8 lock nucleotide.Lock nucleotide can have 2 ' to 4 ' bridge construction described in WO 2012/083005 (mode is in full incorporated herein).Such as, lock nucleotide and can have 2 ' to the 4 ' bridge comprising vinyl or methylene.In other embodiments, lock nucleotide and can have 2 ' to 4 ' methylene bridge.
Generally speaking, the quantity of the length of oligonucleotide and lock nucleotide and position be make this oligonucleotide in vitro in real-time fluorescence quantitative PCR analysis or Luciferase Assay under the oligonucleotide concentration of about 50nM or lower, or to be applicable to as described herein in rodent model or non-human primate model, at about 25mg/kg or more under low dosage, reducing the activity of one or more miR-15 family member.In one embodiment, the length of oligonucleotide is about 8 to about 18 nucleotide.In another embodiment, the length of oligonucleotide is about 12 to about 17 nucleotide.
In an exemplary embodiment, the length of oligonucleotide is about 16 nucleotide.Oligonucleotide can comprise and is selected from following nucleotide sequence or substantially forms by being selected from following nucleotide sequence: 5 '-ACCATTATGTGCTGCT-3 ' (SEQ ID NO.1), 5 '-ACCATGATGTGCTGCT-3 ' (SEQ ID NO.2), 5 '-ATATTTACGTGCTGCT-3 ' (SEQ ID NO.3), and 5 '-ATATTTCTGTGCTGCT-3 ' (SEQ ID NO.4).Oligonucleotide can comprise 9 lock nucleotide or 7 non-lock nucleotide.The pattern of lock nucleotide can be lock nucleotide at least position 1,5,8,10 and 16.
In another exemplary embodiment, the length of oligonucleotide is about 12 nucleotide.Oligonucleotide can comprise and is selected from following nucleotide sequence or substantially forms by being selected from following nucleotide sequence: 5 '-TTATGTGCTGCT-3 ' (SEQ ID NO.5), 5 '-TTACGTGCTGCT-3 ' (SEQ ID NO.6), 5 '-TTCTGTGCTGCT-3 ' (SEQ ID NO.7), 5 '-TTCCGTGCTGCT-3 ' (SEQ IDNO.8), 5 '-TGATGTGCTGCT-3 ' (SEQ ID NO.9), 5 '-TGACGTGCTGCT-3 ' (SEQ ID NO.10), 5 '-TGCTGTGCTGCT-3 ' (SEQ IDNO.11), with 5 '-TGCCGTGCTGCT-3 ' (SEQ ID NO.12).Oligonucleotide can comprise 8 lock nucleotide and 4 non-lock nucleotide.The pattern of lock nucleotide can be at least position Isosorbide-5-Nitrae, and 9 and 12 is lock nucleotide.
In another exemplary, the length of oligonucleotide is about 8 nucleotide.Oligonucleotide can substantially by or be made up of sequence 5 '-GTGCTGCT-3 ', wherein all or substantially all (such as, at least 6 or at least 7) are lock nucleotide.
In each embodiment, comprise with the region of the seed zone complementation of one or more miR-15 family member and lock nucleotide at least about 4, in another embodiment, oligonucleotide is containing having non-lock nucleotide continuous in 3 and/or the continuous nucleotide section of locking nucleotide more than 3.
About non-lock nucleotide, nucleotide can be modified containing about 2 ' of 2 ' hydroxyl.In some embodiments, 2 ' modification can independently selected from deoxy, O-alkyl, O-methyl, halogen, and fluorine-based.Such as, 2 ' modification can be deoxy.In one embodiment, all in oligonucleotide non-lock nucleotide is 2 ' deoxy.
Oligonucleotide also can comprise one or more thiophosphate and connect.Such as, oligonucleotide can be maybe can connecting containing the thiophosphate of have an appointment half or 3/4 of complete thiophosphate connection.
Exemplary oligonucleotide inhibitor is shown in Table 1.
In other embodiments, oligonucleotide can containing 5 ' and or 3 ' cap.In yet another embodiment, oligonucleotide can comprise the lipophilic group (pendent lipophilic group) of side joint further.
On the other hand, the invention provides the pharmaceutical compositions and preparaton that comprise oligonucleotide of the present invention, it can relate in multiple macromolecular assemblies (macromolecular assembly), micelle or the liposome composition being incorporated into by oligonucleotide and sending for cell.In certain embodiments, oligonucleotide preparation is used for traditional intravenous, subcutaneous or intramuscular dose administration.Such preparaton can be traditional aqueous formulation, such as saline agent.In certain embodiments, compositions is applicable to or is formulated as Intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection, or direct injection is to target tissue (such as, heart tissue).
In other respects, the invention provides a kind of for by oligonucleotide and pharmaceutical compositions in vitro or ex vivo delivered to the method for mammalian cell, such as, be used for the treatment of, improve or prevent the progress of the situation of mammalian subject.Oligonucleotide or the compositions that comprises this oligonucleotide are used targeted cell population or mammalian subject by the effective dose that the method can comprise Pim1 target thing is disinthibited.Experimenter can have relevant to the expression of one or more miR-15 family member, the situation being mediated by the expression of one or more miR-15 family member or caused by the expression of one or more miR-15 family member.Such as, this kind of condition of illness comprises cardiac hypertrophy, myocardial infarction, heart failure (such as, congestive heart failure), ischemia, ischemical reperfusion injury, blood vessel injury, restenosis, pathological heart fibrosis or the situation relevant to heart transplantation.Therefore, the invention provides a kind of modified oligonucleotides and compositions is used for the treatment of these situations and the purposes for the preparation of the medicine for the treatment of for these.
Other aspects of the present invention and embodiment will be described in detail below of the present invention and apparent.
Accompanying drawing explanation
Fig. 1. the abundance of miR-15 family member in myocardial cell.In each cell, the copy number of Microrna is measured by real-time PCR analysis, and relative to commercial standard (Ambion) standardization.
Fig. 2. miR-15 target thing in heart tissue.Real-time PCR analysis shows, Bcl2L2, Birc5, Grn in the anti-miR15b of subcutaneous delivery 25mg/kg (M-10134) inducing mouse, and firm (robust) change that Cdc2A expresses.
Fig. 3. anti-miR15b (M-10134) treatment affects expression of target gene with dosage-dependent manner.Real-time PCR analysis shows miR-15 target thing in mice, especially Birc5, Grn, and the dose dependent of Cdc2A disinthibites (de-repreesion).M-10591 is non-target tropism control oligonucleotide.
Fig. 4. the effect of the miR-15 inhibitor for miR-15 target thing measured by real-time PCR analysis.Inhibitor M-10670, M-11211, M-11213, M-11214, M-11215, M-11220, M-11221, and M-11222 seems to play the strongest effect to miR-15 target gene in mice.Error bar describes SEM.The group of P value <0.001 represents with asterisk.
Fig. 5. to use real-time PCR analysis quantitatively inhibitor activity in vitro
microrna analyzes (Applied Biosystems).
Fig. 6. Fig. 6 A shows by for miR-15a, miR-15b, miR-16, and miR-15 inhibitor effect that the two step real-time PCR analysis of miR-195 are measured.Fig. 6 B is the chart of effect of each miR-15 inhibitor of general introduction.
Fig. 7. the psiCHECK of the two quantitative inhibitor activity of Luciferase Assay of external use
tM-2 constructs (Promega).
Fig. 8. Fig. 8 A shows by for miR-15a, miR-15b, miR-16, and miR-15 inhibitor effect of two Luciferase Assay measurements of miR-195.Fig. 8 B is the chart of effect of each miR-15 inhibitor of general introduction.
Fig. 9. effect that one group of miR-15 inhibitor disinthibites to target gene.Fig. 9 A shows, and the therapeutic scheme inducing mouse target gene of 3 doses of (every agent 25mg/kg) miR-15 inhibitor disinthibites.Have and specify the statistically significant group of p value to represent with asterisk.Fig. 9 B shows, and the therapeutic scheme of the miR-15 inhibitor of 3 doses (every agent 25mg/kg) is more effective than single dose regimen (every agent 25mg/kg).
The kinetics that Figure 10 .miR-15 inhibitor makes target gene disinthibite.Effective silence of single dose (25mg/kg) M-11215 early 24 hours inducing mouse miR-15 target things is after injection sent in Figure 10 display.
Figure 11. disinthibited by the overall situation of miR-15 inhibitor and specific target gene.Figure 11 shows, and M-11214 causes target gene to disinthibite, and it has specificity (p-value: 0.01) to miR-15 family.Hypergeometric distribution function is used to calculate the p value of enrichment.
Figure 12. effect that one group of miR-15 inhibitor disinthibites to rat target gene.Figure 12 A shows, and subcutaneous injection single dose miR-15 inhibitor (25mg/kg) after the treatment four days induction target genes disinthibites.In rats, 16 aggressiveness inhibitor seem more effective than 12 aggressiveness inhibitor.Figure 12 B more after the treatment the 2nd day miR-15 inhibitor in rat with the removal of inhibit function for mice target gene.The data of left figure result from rat, and the data of right figure result from mice.Generally speaking, miR-15 inhibitor seems in rats than having more effective effect in mice, but except 12 aggressiveness inhibitor, it seems invalid in rats.
Figure 13. effect that miR-15 inhibitor disinthibites to target gene in stress in rats model.Figure 13 shows, and single dose miR-15 inhibitor (25mg/kg) induces target gene to disinthibite (n=4 in ischemia-reperfusion model; Relative to baseline p value ﹤ 0.05).P value uses ANOVA Newman-Ke Yier post-hoc tests (Newman-Keuls post test) to calculate.Baseline represents non-infraction matched group.IR/ saline represents the matched group of saline treatment.M-10591 is non-target tropism control oligonucleotide.
Figure 14. effect that in stress in rats model, miR-15 inhibitor is expressed Markers of inflammation.Figure 14 shows single dose miR-15 inhibitor (25mg/kg) in ischemia-reperfusion model to the impact (n=4) that various Markers of inflammation is expressed.Baseline represents non-infraction matched group.IR/ saline represents the matched group of saline treatment.M-10591 is non-target tropism control oligonucleotide.
Figure 15. in stress in rats model, miR-15 inhibitor is to effect of myocardial infarction.Figure 15 A and Figure 15 B show the impact (n=10) on the size of risk area (area ofrisk) and myocardial infarction in ischemia-reperfusion model of miR-15 inhibitor.Saline represents the matched group of saline treatment.M-10591 is non-target tropism control oligonucleotide.
Figure 16. in stress in rats model, miR-15 inhibitor (M-11211 and M-11214) is to effect of ejection fraction.Figure 16 shows the impact on ejection fraction in ischemia-reperfusion model of miR-15 inhibitor.AMC represents undamaged age matched control group.IR/ saline represents the contrast of saline treatment.
Detailed Description Of The Invention
The invention provides and can suppress to comprise miR-15a, miR-15b, miR-16, miR-195, miR-424, and the oligonucleotide of the chemical modification of the expression (such as abundance) of the miR-15 family miRNAs of miR-497.The present invention provides in some embodiments and can suppress miR-15a in a specific way, miR-15b, miR-16, miR-195, miR-424, and the oligonucleotide of expression in miR-497 each or abundance.Invention further provides the pharmaceutical compositions comprising oligonucleotide, and treatment suffers from or relates to the condition of illness of miR-15 family miRNA or the method for the disease such as patient of various cardiovascular status relevant.In each embodiment, in one or more in effect, delivery efficiency, target-specific, toxicity and/or stability of oligonucleotide, provide benefit.
On the one hand, the invention provides and can reduce the expression of miR-15 family miRNAs or the oligonucleotide of abundance.The activity of oligonucleotide can in vitro and/or in vivoassay.Such as, when measuring miR-15a in vitro, miR-15b, miR-16, miR-195, miR-424, and during the suppression of miR-497 activity, activity can use two step real-time PCR analysis described herein or two Luciferase Assay to measure.As in such as two Luciferase Assay measure, oligonucleotide is at about 75nM or lower, or about 50nM or lower, about 40nM or lower, about 20nM or lower in other embodiments, or significantly suppresses this activity under the concentration of about 10nM or lower.Such as, oligonucleotide can have about 50nM or lower, about 40nM or lower, about 30nM or lower, or the suppression miR-15a of about 20nM or lower, miR-15b, miR-16, miR-195, miR-424, and the IC50 value of miR-497 activity.
As commercially available
microRNA analyzes exemplified by (Applied Biosystems), two-step pcr analysis relates to quantitative PCR and reads, and it suppresses with two steps by oligonucleotide inhibitor: the ability 1) by suppressing reverse transcriptase primer to extend in cDNA synthesis; With 2) by the ability of suppression PCR primer amplification cDNA product.
As commercially available product P siCHECK
tM(Promega), exemplified by, two Luciferase Assay relates to puts miR recognition site in the 3 ' UTR that can detect albumen (such as Rluc) gene.Construct and target miRNA coexpression, thus measure inhibitor activity by the change of signal.The second gene that coding can detect albumen (such as Fluc) can be included on same plasmid and measured signal than the instruction as anti-miR activity.
Or, or in addition, the activity of oligonucleotide can measure in the mice be applicable to as described in this article or rat model, wherein at about 50mg/kg or lower, about 25mg/kg or lower, all 10mg/kg according to appointment or lower, or under the oligonucleotide dosage of about 5mg/kg or lower, observe the suppression (such as, at least about 50%) to miR-15 family miRNA.In some embodiments, in the animal model described by WO2008/016924 (being incorporated herein by reference), oligonucleotide activity is measured.Such as, oligonucleotide can at about 50mg/kg or lower, about 25mg/kg or lower, all 10mg/kg according to appointment or lower, or represent under the dosage of about 5mg/kg or lower at least about 50% target miRNA suppress or target disinthibite.In this kind of embodiment, oligonucleotide can through intravenous or subcutaneous to rat or mice administration, and oligonucleotide is prepared in saline.
In these or other embodiment, oligonucleotide of the present invention is stable after administration, upon administration at least about three weeks, at least about surrounding, at least about five weeks or can detect at least about in six weeks or longer time inherent circulation and/or target organ.Therefore, oligonucleotide of the present invention has provides administration not too frequently, compared with low dosage and/or the potential treating acting duration compared with Changzhi.
Oligonucleotide comprise nucleotide sequence 5 '-GTGCTGCT-3 ' and with people miR-15a, miR-15b, miR-16, miR-195, miR-424, and the nucleotide sequence of miR-497 is complementary in fact.Oligonucleotide is further containing at least one lock nucleotide.Such as, oligonucleotide can containing at least 8 or at least 9 lock nucleotide.Generally speaking, the number of the length of oligonucleotide and lock nucleotide and position make oligonucleotide in vitro in Luciferase Assay under the oligonucleotide concentration of about 50nM or lower, or at about 50mg/kg or lower in applicable mice or rat model, or under the dosage of about 25mg/kg or lower (as described herein), reduce miR-15a, miR-15b, miR-16, miR-195, miR-424, and the activity of miR-497.The oligonucleotide of complementation is relative to its target sequence miR-15a in fact, miR-15b, miR-16, miR-195, miR-424, and miR-497 can have about 1 to about 5 mispairing (such as, 1 or about 2, or about 3, or about 4, or about 5 mispairing).
Describe in WO 2009/062169 (it is incorporated herein by reference) and comprise miR-15a, miR-15b, miR-16, miR-195, miR-424, with structure and the processing of the miR-15 family member of miR-497, and they are used for the treatment of the potentiality of cardiac hypertrophy, heart failure or myocardial infarction (in other symptoms).The miRNA precursor sequence (such as stem ring sequence) that can be used for the people miR-15 family member designing inhibition miRNAs according to the present invention is listed in hereinafter:
People miR-15a precursor
CCUUGGAGUA AAGUAGCAGC ACAUAAUGGU UUGUGGAUUUUGAAAAGGUG CAGGCCAUAU UGUGCUGCCU CAAAAAUACA AGG(SEQ ID NO.13)
People miR-15b precursor
UUGAGGCCUU AAAGUACUGU AGCAGCACAU CAUGGUUUACAUGCUACAGU CAAGAUGCGA AUCAUUAUUU GCUGCUCUAGAAAUUUAAGG AAAUUCAU(SEQ ID NO.14)
People miR-16-1 precursor
GUCAGCAGUG CCUUAGCAGC ACGUAAAUAU UGGCGUUAAGAUUCUAAAAU UAUCUCCAGU AUUAACUGUG CUGCUGAAGUAAGGUUGAC(SEQ ID NO.15)
People miR-16-2 precursor
GUUCCACUCU AGCAGCACGU AAAUAUUGGC GUAGUGAAAUAUAUAUUAAA CACCAAUAUU ACUGUGCUGC UUUAGUGUGA C(SEQID NO.16)
People miR-195 precursor
AGCUUCCCUG GCUCUAGCAG CACAGAAAUA UUGGCACAGGGAAGCGAGUC UGCCAAUAUU GGCUGUGCUG CUCCAGGCAGGGUGGUG(SEQ ID NO.17)
People miR-424 precursor
CGAGGGGAUA CAGCAGCAAU UCAUGUUUUG AAGUGUUCUAAAUGGUUCAA AACGUGAGGC GCUGCUAUAC CCCCUCGUGGGGAAGGUAGA AGGUGGGG(SEQ ID NO.18)
People miR-497 precursor
CCACCCCGGU CCUGCUCCCG CCCCAGCAGC ACACUGUGGUUUGUACGGCA CUGUGGCCAC GUCCAAACCA CACUGUGGUGUUAGAGCGAG GGUGGGGGAG GCACCGCCGA GG(SEQ ID NO.19)
The miRNA precursor sequence of each miR-15 family member is processed as mature sequence and star sequence separately.Star sequence is processed from another chain of loop-stem structure.Mature sequence and the star sequence of each miR-15 family member provide as follows:
The ripe miR-15a of people
UAGCAGCACAUAAUGGUUUGUG(SEQ ID NO.20)
People miR-15a*
CAGGCCAUAUUGUGCUGCCUCA(SEQ ID NO.21)
The ripe miR-15b of people
UAGCAGCACAUCAUGGUUUACA(SEQ ID NO.22)
People miR-15b*
CGAAUCAUUAUUUGCUGCUCUA(SEQ ID NO.23)
The ripe miR-16-1/miR-16-2 of people
UAGCAGCACGUAAAUAUUGGCG(SEQ ID NO.24)
People miR-16-1*
CCAGUAUUAACUGUGCUGCUGA(SEQ ID NO.25)
People miR-16-2*
CCAAUAUUACUGUGCUGCUUUA(SEQ ID NO.26)
The ripe miR-195 of people
UAGCAGCACAGAAAUAUUGGC(SEQ ID NO.27)
People miR-195*
CCAAUAUUGGCUGUGCUGCUCC(SEQ ID NO.28)
The ripe miR-424 of people
CAGCAGCAAUUCAUGUUUUGAA(SEQ ID NO.29)
People miR-424*
CAAAACGUGAGGCGCUGCUAU(SEQ ID NO.30)
The ripe miR-497 of people
CAGCAGCACACUGUGGUUUGU(SEQ ID NO.31)
People miR-497*
CAAACCACACUGUGGUGUUAGA(SEQ ID NO.32)
Although the seed zone of all family members (such as, crossing over 2 bases to 8 nucleotide of ripe miRNA sequence) is high conservative (AGCAGCAC), the 3 ' end of the ripe miRNA between different family member is different.
Expect by giving single miR-15 inhibitor or give multiple members that various inhibitors suppresses miR-15 family simultaneously.Such as, each inhibitor can targeting single miR-15 family member or can the multiple miR-15 family member of targeting.In some embodiments, single miR-15 inhibitor suppresses expression or the activity of two or more miR-15 family member.This kind of inhibitor includes, but are not limited to: M-10670, M-11211, M-11213, M-11214, M-11215, M-11220, M-11221, and M-10222.In other embodiments, single miR-15 inhibitor suppresses expression or the activity of more than three miR-15 family members.This kind of inhibitor includes, but are not limited to: M-10670, M-11211, M-11213, M-11214, M-11215, M-11220, M-11221, and M-10222.In other embodiments, single miR-15 inhibitor suppresses expression or the activity of four or more miR-15 family member.This kind of inhibitor can include, but are not limited to: M-11211, M-11213, M-11214, M-11215, M-11220, and M-11221.
Oligonucleotide contains one or more lock nucleotide (LNA) residue, or " lock nucleotide ".Such as LNA is recorded in United States Patent (USP) the 6th, 268, No. 490, United States Patent (USP) the 6th, 316, No. 198, United States Patent (USP) the 6th, 403, No. 566, United States Patent (USP) the 6th, 770, No. 748, United States Patent (USP) the 6th, 998, No. 484, United States Patent (USP) the 6th, 670, No. 461, and United States Patent (USP) the 7th, 034, No. 133, its mode quoted in full is incorporated herein.LNA be containing ribose moieties 2 ' with 4 ' between carbon containing extra bridge, produce " lock " thus and construct, and/or the modified nucleotide of twin nuclei and ribonucleotide.In some embodiments, oligonucleotide contains and one or morely has the LNA shown in following structure A.Or or in addition, oligonucleotide can have the LNA shown in following structure B containing one or more.Or or in addition, oligonucleotide contains and one or morely has the LNA shown in following structure C.
Other suitable lock nucleotide that can be incorporated in oligonucleotide of the present invention comprise these and are recorded in U.S. Patent number the 6th, and the lock nucleotide of 403, No. 566 and U.S. Patent number the 6th, 833,361, these modes quoted all are in full incorporated herein.
In an exemplary embodiment, lock nucleotide and there is 2 ' to 4 ' methylene bridge, such as, as shown in structural formula A.In other embodiments, lock nucleotide has the bridge comprising vinyl, and it can contain in 2 ' position or not contain ehter bond.
Oligonucleotide can comprise, form and be essentially or consist of the total length of miR-15 family member or the antisense sequences of truncate.As used in this article, the length of ripe miRNA antisense homologue (counterpart) is referred to about the term " total length " of miRNA sequence.Therefore, the ripe miRNA sequence of antisense that is that inhibitor described herein can be truncate or total length, maybe can comprise the combination of these sequences and other polynucleotide sequences.In these embodiments, described herein chemical modification primitive makes total length antisense miRNA (maturation) sequence become unnecessary.In these embodiments, oligonucleotide is that about 8 to about 20 nucleotide are long, or is that about 8 to about 18 nucleotide are long, or is that about 12 to 17 nucleotide are long.In some embodiments, oligonucleotide is that about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16 nucleotide are long.The oligonucleotide of truncate can have the sequence being carried out the following sequence of targeting by Antisense Suppression: the miR-15a sequence in 5 '-UAGCAGCACAUAAUGGU-3 ' (SEQ ID NO.33), miR-15b sequence in 5 '-UAGCAGCACAUCAUGGU-3 ' (SEQ ID NO.34), miR-16 sequence in 5 '-UAGCAGCACGUAAAUAU-3 ' (SEQ ID NO.35), miR-195 sequence in UAGCAGCACAGAAAUAU-3 ' (SEQ ID NO.36), miR-424 sequence in 5 '-CAGCAGCAAUUCAUGUU-3 ' (SEQ ID NO.37), or 5 ' miR-497 sequence in-CAGCAGCACACUGUGGU-3 ' (SEQ ID NO.38).
Oligonucleotide usually has and is designed to targeting ripe miR-15a, miR-15b, miR-16, miR-195, miR-424, and the nucleotide sequence of miR-497.In these or other embodiment, oligonucleotide also or can be designed to targeting miRNA precursor or primary transcribe (pri-miRNA) form.In certain embodiments, oligonucleotide can be designed to have the sequence comprising 1 to 5 (such as, about 1, about 2, about 3, about 4) mispairing of having an appointment relative to the miR-15 sequence of total length complementation (ripe).In certain embodiments, these antisense sequences can be incorporated into as shRNAs or other contain in the RNA structure of stem and loop section.
In an exemplary embodiment, oligonucleotide is that about 16 nucleotide are long.Oligonucleotide can comprise and is selected from following nucleotide sequence or substantially forms by being selected from following nucleotide sequence: 5 '-ACCATTATGTGCTGCT-3 ' (SEQ ID NO.1), 5 '-ACCATGATGTGCTGCT-3 ' (SEQ ID NO.2), 5 '-ATATTTACGTGCTGCT-3 ' (SEQ ID NO.3), and 5 '-ATATTTCTGTGCTGCT-3 ' (SEQ ID NO.4).Oligonucleotide can comprise the mixture of lock nucleotide and non-lock nucleotide.Such as, oligonucleotide can containing 9 lock nucleotide and 7 non-lock nucleotide.The pattern of lock nucleotide can make at least position 1,5,8,10 and 16 be lock nucleotide.
In the embodiment that another is exemplary, oligonucleotide is that about 12 nucleotide are long.Oligonucleotide can comprise following nucleotide sequence or substantially formed by being selected from following nucleotide sequence: 5 '-TTATGTGCTGCT-3 ' (SEQ ID NO.5), 5 '-TTACGTGCTGCT-3 ' (SEQ ID NO.6), 5 '-TTCTGTGCTGCT-3 ' (SEQ ID NO.7), 5 '-TTCCGTGCTGCT-3 ' (SEQ IDNO.8), 5 '-TGATGTGCTGCT-3 ' (SEQ ID NO.9), 5 '-TGACGTGCTGCT-3 ' (SEQ ID NO.10), 5 '-TGCTGTGCTGCT-3 ' (SEQ ID NO.11), with 5 '-TGCCGTGCTGCT-3 ' (SEQ ID NO.12).Oligonucleotide can comprise the mixture of lock nucleotide and non-lock nucleotide.Such as, oligonucleotide can containing 8 lock nucleotide and 4 non-lock nucleotide.The pattern of lock nucleotide can make at least position 1,4,9 and 12 be lock nucleotide.
In the embodiment that another is exemplary, oligonucleotide is that about 8 nucleotide are long.Oligonucleotide can substantially by, or be made up of nucleotide sequence 5 '-GTGCTGCT-3 ', wherein all or in fact all (such as, at least about 6 or at least about 7) is lock nucleotide.
Oligonucleotide locks nucleotide containing at least about 8 usually, but is not all be made up of lock nucleotide in each embodiment.Usually, the lock quantity of nucleotide and position be make as described in vitro or or in vivoassay time, oligonucleotide reduces miR-15a, miR-15b, miR-16, miR-195, miR-424, and the activity of miR-497.In certain embodiments, oligonucleotide is containing having the nucleotide section exceeding about 4 or exceed about 3 adjacent non-lock nucleotide.In these or other embodiment, with miR-15a, miR-15b, miR-16, miR-195, miR-424, and the region of miR-497 seed zone complementation comprises at least 3 or at least 4 lock nucleotide.
For non-lock nucleotide, nucleotide can be modified containing for 2 ' of 2 ' hydroxyl.Such as 2 ' modification can be 2 ' deoxy.In antisense oligonucleotide, be incorporated to the 2 ' nucleotide modified can increase oligonucleotide to the resistance of nuclease and and the heat stability of complementary RNA thereof.Various modifications in 2 ' position independently can be selected from increase nuclease sensitivity and not damage the synergistic modification of molecule and RNA target or cellular machineries.These modifications can based on it in external or body the increase of effect select.The illustrative methods increasing (such as IC50) for measuring the efficiency suppressed for miRNA is described in herein, comprises miRNA expression or target in two Luciferase Assay and body and disinthibites.
In some embodiments, 2 ' modification can independently selected from O-alkyl (it can be substituted), halogen and deoxy (H).In certain embodiments, 2 ' position of all in fact or all nucleotide of non-lock nucleotide can be modified.Such as, as being independently selected from O-alkyl (such as, O-methyl), halogen (such as, fluorine-based), deoxidation (H) and amino.Such as, 2 ' modify separately can independently selected from O-methyl and fluorine-based.In an exemplary embodiment, purine nucleotides has 2 ' OMe separately, and pyrimidine nucleotide has 2 '-F separately.In some embodiments, 1 to about 52 ' position, or about 1 to about 32 ' position keeps not modified (such as, as 2 ' hydroxyl).
Modify according to of the present invention 2 ' and also comprise little hydrocarbon replacement.Hydrocarbon substituent comprises alkyl, thiazolinyl, alkynyl and alkoxyalkyl, and wherein alkyl (comprising the moieties of alkoxyl), thiazolinyl, alkynyl can be substituted or non-substituted.Alkyl, thiazolinyl, alkynyl can be C1 to C10 alkyl, alkenyl or alkynyl, such as C1, C2 or C3.Hydrocarbon substituent can comprise 1 or 2 or 3 non-carbon, and these non-carbon independently can be selected from N, O, and/or S.2 ' modification can comprise further containing being O-alkyl, O-thiazolinyl, and the alkyl of O-alkynyl, thiazolinyl and alkynyl.
Modify according to of the present invention exemplary 2 ' and comprise 2 '-O-alkyl (C1-3 alkyl, such as 2 ' OMe or 2 ' OEt), 2'-O-methoxy ethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethyl aminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylamino ethoxy ethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamido (2'-O-NMA) replaces.
In certain embodiments, oligonucleotide contains at least one 2 ' halogen to be modified (such as, replacing 2 ' hydroxyl), such as 2 '-fluorine-based, 2 '-chloro, 2 '-bromo and 2 '-iodo.In some embodiments, 2 ' fluorine-based be modified to fluorine-based.Oligonucleotide can modify (such as, fluorine-based) containing 1 to about 52 '-halogen, or 1 to about 32 ' halogen modifies (such as, fluorine-based).In some embodiments, oligonucleotide contains all 2 ' fluorine-based nucleotide at non-lock position place, or on all non-lock pyrimidine nucleotides 2 '-fluorine-based.In certain embodiments, 2 ' fluorine-based for twoly independently to methylate, tri-methylated or do not methylate.
Oligonucleotide can have one or more 2 '-deoxy and modify (such as, hydrogen for 2 ' hydroxyl), and in some embodiments, modify containing 2 to about 10 2 '-deoxies of having an appointment at non-lock position place, or contain 2 ' deoxy at all non-lock position places.
In an exemplary embodiment, oligonucleotide in non-lock position containing the 2 ' position being modified to 2 ' OMe.Or non-lock purine nucleotides is modified to 2 ' OMe in 2 ' position, and non-lock pyrimidine nucleotide be modified to 2 ' in 2 ' position-fluorine-based.
In some embodiment, oligonucleotide comprises at least one end modified or " cap " further.Cap can be 5 ' and/or 3 ' cap.Term " cap " or " cap end " are included in the chemical modification at the arbitrary end of oligonucleotide (relative to terminal ribose sugar nucleotide) place, and to be included on 5 ' end the modification that latter two nucleotide and 3 ' holds junction between latter two nucleotide.Cap as described herein can increase the resistance of oligonucleotide to exonuclease, and does not damage the interaction of molecules of molecule and RNA target or cellular machineries.This kind of modification can based on it in external or body the increase of effect select.Cap can be present in 5 ' end (5 ' cap) or 3 ' end (3 ' cap), maybe can be present on two ends.In certain embodiments, 5 ' and/or 3 ' cap is independently selected from monophosphate thiophosphate, without base residue (part), phosphorothioate bond, 4 '-sulfenyl nucleotide, homocyclic nucleus thuja acid, phosphordithiic acid ester bond, reverse nucleotide or oppositely abasic moiety (2 '-3 or 3 '-3 '), monophosphate phosphorodithioate and methyl orthophosphoric acid part.When thiophosphate or phosphordithiic acid ester bond are cap a part of, its two terminal nucleotide and 3 ' be usually positioned on 5 ' end are held between two terminal nucleotide.
In certain embodiments, oligonucleotide has at least one end monophosphate thiophosphate.Monophosphate thiophosphate by suppress exonuclease be used for support higher effect.Monophosphate thiophosphate can at 5 ' of oligonucleotide and/or 3 ' end place.Monophosphate thiophosphate is by being defined by following structure, and wherein B is base, and R is 2 ' modification as described above:
Cap can support lock core nucleotide chemistry character time, cap can be incorporated in lock nucleotide described herein.
Phosphorothioate bond can be present in some embodiments, such as on 5 ' and 3 ' end between latter two nucleotide (such as, the part as cap sequence), or replaces with phosphodiester bond.In these or other embodiment, oligonucleotide can 5 ' and 3 ' any one of holding or both places containing at least one end without base residue.Abasic moiety is not containing the purine of generally acknowledging or pyrimidine nucleotide base, such as adenosine, guanine, cytosine, uracil or thymus pyrimidine.Therefore, this kind of abasic moiety lacks nucleotide base at 1 ' position place or has other non-nucleotide base chemical groups.Such as, can be antisense without nucleotide base without nucleotide base, such as, wherein reverse is via 5 ' amide (amidite) (substituting 3 ' amide) coupling without base phosphoramidite, produces 5 '-5 ' phosphoric acid ester bond.Below show polynucleotide 5 ' and 3 ' the reverse structure without base nucleosides of holding.
Oligonucleotide can contain one or more phosphorothioate bond.Phosphorothioate bond is for making oligonucleotide have more resistance to nuclease cleavage.Such as, polynucleotide can be that partly thiophosphate connects, and such as, phosphorothioate bond can replace with phosphodiester bond.But in certain embodiments, oligonucleotide is that complete thiophosphate connects.In other embodiments, oligonucleotide has about 1 to 5 or about 1 to about 3 phosphoric acid ester bond.
In some embodiments, described by WO 2012/061810 (it is incorporated herein by reference), nucleotide has one or more base of modifying through carboxamido (carboxamido), comprises and modifies about all exemplary pyrimidine carboxamido with heterocyclic substituent disclosed in this patent.
In an exemplary embodiment, oligonucleotide has the structure of compound listed in following table 1.Described by table 1, plus sige (such as ,+A) represents LNA base.Other bases all are DNA.Each miR-15 inhibitor has the main chain that complete thiophosphate connects.
table 1: exemplary oligonucleotide
The oligonucleotide being synthesized the polynucleotide comprising modification by solid phase synthesis is what know, and comments in New Chemical Methods for Synthesizing Polynucleotides.Caruthers MH,Beaucage SL,Efcavitch JW,Fisher EF,Matteucci MD,Stabinsky Y.NucleicAcids Symp.Ser.(7):215-23,(1980)。
Oligonucleotide can be incorporated in multiple macromolecular assemblies or compositions.These can comprise through preparation for delivery to the multiple liposome of patient, nanoparticle and micelle for the complex sent.Complex can comprise one or more short melting property (fusogenic) or lipophilic molecules penetrates with initiator cell film.These molecules are recorded in such as United States Patent (USP) the 7th, 404,969 and No. the 7th, 202,227, United States Patent (USP), and it is incorporated herein by the mode quoted in full.Or oligonucleotide can comprise side joint lipophilic group further and send with accessory cell, the group recorded in such as WO 2010/129672 (it is incorporated to herein by reference).
Compositions and formulation can use multiple therapeutic oligonucleotide, comprise described at least one herein.Such as, compositions and formulation can use at least about 2, about 3, about 4 or about 5 described herein miRNA inhibitor.
Oligonucleotide of the present invention can be formulated as multi-medicament compositions.Pharmaceutical composition is by the form preparation to be suitable for predetermined application.Usually, this compositions that will preparation needed to be substantially free of thermal source matter and other impurity to the mankind or animal pest.Exemplary delivery/compounding system comprises colloidal dispersion systems, macromolecular complex, nanocapsule, microsphere, beadlet, and comprises the system based on lipid of O/w emulsion, micelle, mixed micelle and liposome.The commercially available fats emulsion of delivery of nucleic acids of the present invention to heart and skeletal muscle tissue is suitable for comprise
iII, Nutrilipid, and other similar butterfat emulsions.Preferably colloidal system for means of delivery in body is liposome (that is, artificial membrane vesicle).The preparation of this type systematic and using in the art for knowing.Exemplary formulation is also disclosed in United States Patent (USP) the 6th, 217, No. 900; United States Patent (USP) the 6th, 383, No. 512; United States Patent (USP) the 5th, 783, No. 565; United States Patent (USP) the 7th, 202, No. 227; United States Patent (USP) the 6th, 379, No. 965; United States Patent (USP) the 6th, 127, No. 170; United States Patent (USP) the 5th, 837, No. 533; United States Patent (USP) the 6th, 747, No. 014; With WO 2003/093449, it is incorporated herein by the mode quoted in full.
In some embodiments, oligonucleotide is formulated for usual subcutaneous or intravenous administration, such as, by preparing by the suitable aqueous diluent comprising sterilized water and saline.
Pharmaceutical composition and formulation can use suitable salt and buffer to make means of delivery stable and can be absorbed by target cell.Waterborne compositions of the present invention comprises the means of delivery (such as liposome or other complex) comprising inhibitor oligonucleotide of the effective dose be dissolved or dispersed in pharmaceutically acceptable carrier or aqueous medium.Term " pharmaceutically acceptable " or " pharmacologically can accept " refer to that molecular entity and compositions can not produce untoward reaction when giving to animals or humans, anaphylaxis or other improper reactions.As used herein, " pharmaceutically acceptable carrier " can comprise at compounding pharmaceutical, is such as suitable for using one or more solvents acceptable, buffer, solution, disperse medium, coating, antibacterial and antifungal, isotonic agent and absorption delay agent etc. in the medicine given to the mankind.This kind of medium and the reagent use in pharmaceutically active substances is in the art for knowing.Complementarity active component also can be incorporated in compositions.
Target tissue gives or sends to carry out through any approach according to pharmaceutical compositions of the present invention, as long as can utilize through this approach.Such as, by Intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection, or administration is carried out by direct injection to (such as, heart tissue) in target tissue.The stability of oligonucleotide disclosed herein and/or effect allow route of administration easily, comprise subcutaneous, Intradermal and intramuscular.In order to by therapeutic agent delivery to heart, the pharmaceutical compositions comprising miRNA inhibitor also can be given by conduit system or the system being separated coronary circulation.For what therapeutic agent delivery to the various conduit systems of heart and coronary vasodilator were known in the art.Some non-limitative examples being applicable to the delivering method based on conduit in the present invention or arteria coronaria separation method are disclosed in United States Patent (USP) the 6th, 416, No. 510; United States Patent (USP) the 6th, 716, No. 196; United States Patent (USP) the 6th, 953, No. 466, WO 2005/082440, WO 2006/089340, No. 2007/0203445th, U.S. Patent Publication No., No. 2006/0148742nd, U.S. Patent Publication No., and No. 2007/0060907th, U.S. Patent Publication No., its mode quoted all is in full incorporated herein.
Compositions and formulation also can parenteral or intraperitoneal give.For example, can prepare in the water being suitably mixed with surfactant (such as hydroxypropyl cellulose) in free alkali or the solution of the pharmacologically conjugate of acceptable salt.Also and dispersion liquid can be prepared in glycerol, liquid polyethylene glycol and its mixture in oil.Under the generic condition stored and use, these preparations contain antiseptic usually to prevent growth of microorganism.
Be applicable to inject and use or the pharmaceutical form of catheter delivery comprises such as aseptic aqueous solution or dispersion liquid and the sterilized powder for extemporaneous preparation of sterile Injectable solution or dispersion liquid.Usually, these preparations be aseptic and its mobility reach be easy to inject degree.Preparation should be stable under conditions of manufacture and storage, and should provide anticorrosion for the contamination of microorganism (as antibacterial and fungus).The mixture that suitable solvent or disperse medium can contain such as water, ethanol, polyhydric alcohol (such as, glycerol, propylene glycol and liquid polyethylene glycol, and analog) and be applicable to, and vegetable oil.Can such as pass through by use coating, as lecithin; Desired particle size is maintained whereby in dispersion liquid situation; Adequate liquidity is maintained with by use surfactant.The effect of microorganism is prevented by various antibacterial and antifungal (such as, p-Hydroxybenzoate (parabens), methaform, phenol, sorbic acid, thimerosal and analog).In many cases, the isotonic agent comprising such as sugar and sodium chloride is preferably.By the absorption using absorption delay agent (such as, aluminum monostearate and gelatin) to extend Injectable composition in the composition.
Sterile injectable solution is prepared by being incorporated into by appropriate conjugate in the solvent with other compositions required (such as, act as listed above).Usually, dispersion liquid is prepared by being incorporated to by the active ingredient of various sterilizing in the sterile vehicle containing basic dispersion medium and other compositions required (such as, as above cited).When the sterile powders for the preparation of sterile injectable solution, preferably preparation method comprises vacuum drying and Freeze Drying Technique, and it produces the powder of active component and other compositions expected from its previous aseptic filtration liquid.
After the slurries were formulated, solution preferably to give to treat effectively to measure in the mode compatible with Dosage formulations.These preparatons can so that give with multiple dosage form (such as Injectable solution, drug release capsules and analog).Such as, for aqueous solution form parenteral, solution usually through suitably buffering and liquid diluent first such as become with enough saline or glucose to wait and open.This kind of aqueous solution can give with such as intravenous, intramuscular, subcutaneous and intraperitoneal.Optimally, sterile aqueous media well-known to those skilled in the art is adopted, especially according to the present invention.For example, single dose can be dissolved in a NaCl solution such as 1ml, and add in 1000ml hypodermoclysis fluid or be injected in proposed infusion part (see such as, " Remington's Pharmaceutical Sciences " the 15th edition, 1035-1038 and 1570-1580 page).Depend on treat individual condition of illness, some doses change will inevitably be there are.Under any circumstance, be responsible for administration personnel and will determine the suitable dosage of individual one.In addition, for mankind's administration, preparation should meet aseptic, pyrogenicity, overall security and purity rubric required by FDA biological preparation standard office room (FDA Office ofBiologics standards).
The invention provides a kind of for by oligonucleotide delivery to mammalian cell method (such as, a part as compositions described herein or formulation), and be used for the treatment of, improve or prevent the method for progress of condition of illness of mammalian subject.Oligonucleotide or pharmaceutical compositions contact with target cell (such as, mammalian cell) in vitro or in body.Cell can be heart cell.
The method usually comprises and gives oligonucleotide to mammalian subject or target cell group or comprise the compositions of this oligonucleotide.Oligonucleotide as recorded is miRNA inhibitor (such as, having through design with the nucleotide sequence of the expression or activity that suppress miR-15 family miRNA).Therefore, patient can suffer from and expresses condition of illness that is relevant, that mediated by it or caused by it to miR-15 family.These condition of illness are recorded in WO 2009/062169, and it is incorporated herein by reference.These condition of illness comprise such as cardiac hypertrophy, myocardial infarction, heart failure (such as, congestive heart failure), blood vessel injury, ischemia, ischemical reperfusion injury, restenosis or pathological heart fibrosis, and the condition of illness relevant to heart transplantation.Therefore, the invention provides the purposes of modified oligonucleotides of the present invention and compositions, it is used for the treatment of this kind of condition of illness and for the preparation of the medicine for the treatment of for these.
In certain embodiments, patient (such as, human patients) there are one or more risk factor, comprise such as long-standing uncontrolled hypertension, the valvular heart disease of not curing, chronic angina, recent myocardial infarction, congestive heart failure, congenital heart disease tendentiousness and pathologic hypertrophy.Or or in addition, patient can be diagnosed as the genetic predisposition with such as cardiac hypertrophy, maybe can have the family history of such as cardiac hypertrophy.
In this respect, the inhibitor giving miR-15 family member makes one or more symptoms in individual such as cardiac hypertrophy, heart failure, ischemia, ischemical reperfusion injury or myocardial infarction improve, or makes cardiac hypertrophy to the transformation delay of heart failure.One or more symptoms improved can be the motor capacitys such as increased, the cardiac ejection amount increased, the Left ventricular end diastolic pressure reduced, the pulmonary capillary wedge pressure reduced, the cardiac output increased, what increase feels concerned about number (increased cardiac index), the pulmonary artery pressure reduced, reduce left ventricular contraction and diastasis size, the left ventricle reduced and right ventricle wall stress, the wall tension force reduced, the quality of life increased and the disease related morbidity rate of reduction or mortality rate.In addition, the related symptoms using the inhibitor of miR-15 family member can prevent cardiac hypertrophy and be caused by it.
In some embodiments of the present invention, the inhibitor of miR-15 family member can give with other treatment mode combinations.Such as, miR-15 inhibitor of the present invention can be combined with the curative of other types and given, as antihyperlipoproteinemic, arteriosclerosis medicine, antithrombotic/fibrinolysis, blood coagulation medicine, antiarrhythmics, antihypertensive, the curative of congestive heart failure, anti-anginal drug, antimicrobial drug, vasodilator, hormone antagonist, cardiac tonic, diuretic, endothelin receptor antagonists, calcium channel blocking agent, phosphodiesterase inhibitor, Angiotensin II (ACE) converting enzyme inhibitor, cytokine blocking agent/depressant, HDAC depressant or its combination.Combination treatment also can participate in suppressing other miRNA involved in cardiac remodeling to express or activity, other miRNA is as miR-499, miR-208, miR-208b and miR-21, it is recorded in WO 2012/083005 and WO2009/058818, and content is incorporated herein by reference.In addition, miR-15 inhibitor can be combined with operation and given.MiR-15 inhibitor also can give together with non-pharmacological therapeutic modality.Such as, with regard to heart disease, non-pharmacological treatment can relate to the sodium reduced in meals.
In each embodiment, pharmaceutical composition is by parenteral or by giving in direct injection to heart tissue.Parenteral can be intravenous, subcutaneous or intramuscular adminstration.In some embodiments, compositions is given by per os, percutaneous, sustained release, Co ntrolled release, delayed release, suppository, conduit or sublingual administration.In certain embodiments, oligonucleotide is with about 25mg/kg or lower, or about 10mg/kg or lower, or the dosage of about 5mg/kg or lower gives.In these embodiments, oligonucleotide or compositions give by intramuscular or subcutaneous injection or intravenous.
In certain embodiments, in heart tissue or as in patients serum measure, miR-15a, miR-15b, miR-16, miR-195, miR-424, and miR-497 activity reduce or suppressed.
In one embodiment, method of the present invention can comprise the miR-15 family's group inhibitor giving the dosage effectively making Pim1 target thing disinthibite to target cell group or mammalian subject, and this inhibitor can be oligonucleotide described herein.In another embodiment, the present invention also provides the method for the expression regulating Pim1 and other target genes in cell (as Bcl2L2, Birc5, Grn, and Cdc2A), and the method comprises makes cell contact with miR-15 inhibitor.In one embodiment, after giving miR-15 inhibitor, Pim1, Bcl2L2, Birc5, Grn, and the expression of Cdc2A increases.Can before the treatment, detect the expression of Pim1 and/or other described label herein to measure therapeutic response after treatments period or treatment.
In some embodiments, method comprises removing after the treatment further or removes miRNA inhibitor.Such as, having can after the treatment to being decayed or stopping the function of inhibitor with the oligonucleotide of the nucleotide sequence of inhibitor complementation.
Further illustrate the present invention by following examples, these embodiments should not be construed as restriction.The mode that the content of all lists of references quoted in the whole text in this application, patent and publication application and accompanying drawing are quoted all is in full incorporated herein for all objects.
Embodiment
realexecute
example 1: the abundance of miR-15 family member in heart tissue
In heart tissue, the abundance of miR-15 family member is assessed by real-time PCR analysis.Particularly, in people (n=6), pig (n=6) and mice (n=30) myocardial cell, whole RNA is extracted.Then use PCR in real time to measure the Microrna copy number of each cell, and carry out standardization relative to commercially available standard substance (Ambion).
It is the miR-15 family member that in heart tissue, abundance is the highest that result (Fig. 1) shows miR-16, in each cell about 10, and 000 copy number.Comprise miR-15a, miR-15b, miR-195, miR-497, miR-424, and other miR-15 family member of miR-322 is with in each cell about 1,000 copy number is expressed.In addition, miR-322 seems only to express in rodent, and miR-424 expression seems to be limited to larger animal (such as, the mankind and pig).
realexecute
example 2: differentiate the gene target thing regulated by miR-15 family member
Synthesis targeting miR-15 family (one group of miRNA inhibitor (single stranded oligonucleotide) of (miR-15a, miR-15b, miR-16, miR-195, miR-497, miR-424, and miR-322).Sequence and modification pattern are shown in table 1.This group comprises the reverse complemental inhibitor of the different lengths of scope in 8 nucleotide to 16 nucleotide.LNA modify number and oligonucleotide in LNA modify position be change.
Research before has identified the latent gene target thing regulated by miR-15 family member.In order to differentiate the specific miR-15 family target thing in heart tissue, to wild type C57BL6 mouse subcutaneous injection saline, or 25mg/kg control oligonucleotide or anti-miR-15b oligonucleotide (M-10134).Treatments period at 3 days gives 3 dosage.24 hr collections heart tissues after injection the last time, and assess expression of target gene by PCR in real time.Result (Fig. 2) shows, Bcl2L2, Birc5, Grn, and the expression of Cdc2A significantly increases after miR-15b suppresses, and shows that these genes regulate by miR-15b family member in heart tissue.
For verifying miR-15 family target thing further, to wild type C57BL6 mouse subcutaneous injection saline, or 2.8mg/kg, 10mg/kg, or 25mg/kg control oligonucleotide or anti-miR-15b oligonucleotide (M-10134).Treatments period at 3 days gives 3 dosage.24 hr collections heart tissues after injection the last time, and assess expression of target gene by PCR in real time.Result (Fig. 3) shows, Birc5, Cdc2A, and Grn suppresses responsive especially for miR-15b.Particularly, the expression response miR-15b of these genes suppresses to increase with dosage-dependent manner impact.
realexecute
example 3: the activity of the miRNA inhibitor of targeting miR-15 family
The ability of miR-15 family member is suppressed to be tested to 25 kinds of miRNA inhibitor (as shown in table 1).
A. miR-15 family member is suppressed by miRNA inhibitor in vivo
In order to measure miR15 inhibitor effect for miR15 expression of target gene, to wild type C57/BL6 mouse subcutaneous injection saline, or 25mg/kg control oligonucleotide or indivedual anti-miR-15b oligonucleotide.Treatments period at 3 days gives 3 dosage.24 hr collections heart tissues after injection the last time, and assess expression of target gene by PCR in real time.Result such as Fig. 4 shows, and length and the LNA pattern of miRNA inhibitor impart different inhibit activities.Especially, inhibitor M-10670, M-11211, M-11213, M-11214, M-11215, M-11220, M-11221, M-11222 affect the expression of miR-15b target gene (as Birc5, Cdc2A, Grn, CcnD1, and CcnD2) consumingly.The length of these inhibitor changes between 12 nucleotide to 16 nucleotide.Wherein, 12 aggressiveness inhibitor M-11211 seem to show the most effective inhibit activities, have impact on the expression of the miR-15 target gene of 4/5ths.
The ability utilizing two step real-time PCR analysis (Applied Biosystems) to assess miRNA inhibitor to suppress indivedual miR-15 family member.Particularly, comprise M-10670 for what previously differentiated, the active compound of M-11211, M-11213, M-11214, M-11215, M-11220, M-11221, M-11222 is to miR-15a, miR-15b, miR16, and the impact that miR-195 expresses is tested.As previously noted, to wild type C57/BL6 mouse subcutaneous injection saline, or 25mg/kg control oligonucleotide or anti-miR-15 oligonucleotide.Treatments period at 3 days gives 3 dosage.Result (Fig. 6 A and 6B) shows, and 12 aggressiveness inhibitor M-11220 are the optimal inhibition agent of test, and suppresses to comprise miR-15a, miR-15b, miR16, and whole 4 miR-15 family members of miR195A.16 aggressiveness inhibitor (that is, M-10670, M-11211, M-11213, M-11214, and M-11215) are suppressing more than three family members in varying degrees.
B. miR-15 family member is suppressed by miRNA inhibitor in vitro
Two Luciferase Assay is utilized to test the activity of miRNA inhibitor in vitro.Particularly, transfection HeLa cell is carried out with the miR-15 inhibitor of variable concentrations and every hole 25ng reporter plasmid.Particularly, as by needing the institute of the transfection inhibitor concentration in 2 to 50nM scope to confirm, compared with 0.31 to the 8.3nM of other constructs, the two Luciferase construct of miR-195 seems not as other constructs sensitivities in Microrna suppression.Result (Fig. 8 A and 8B) shows, and 12 aggressiveness inhibitor M-11220 are tested optimal inhibition agent, and suppress whole 4 miR-15 family members.16 aggressiveness inhibitor (that is, M-10670, M-11211, M-11213, M-11214, and M-11215) are suppressing more than three family members in varying degrees.
realexecute
example 4: the activity in vivo of the miRNA inhibitor of targeting miR-15 family
A.miRNA inhibitor makes miR-15 target thing disinthibite with dosage-dependent manner
Carry out the various dose scheme studying to compare miR-15 inhibitor.Particularly, to the inhibitor M-10670 of one group of mouse subcutaneous injection 25mg/kg dosage, M-11211, M-11213, M-11214, M-11215, M-11220, M-11221, or M-11222.These inhibitor give 3 (3x25MPK scheme) (Fig. 9 A) within the time of 3 days.Comparatively speaking, second group of mice only uses the 25mg/kg miR-15 inhibitor (1x25MPK scheme) of single dose to process.Result (Fig. 9 B) show, 3x25MPK scheme affect in expression of target gene more effective than 1x25MPK scheme.
The kinetics that B.miRNA inhibitor makes target thing disinthibite
Carry out the kinetics that time-histories research makes target thing disinthibite to measure miR-15 inhibitor.Particularly, to mouse subcutaneous injection 25mg/kg M-11215A.Only give single dose.24 hours after injection, 48 hours, 72 hours or 98 hr collections heart tissues.As shown in Figure 10, namely the M-11215 of single dose early causes disinthibiting of target gene (as Birc5) in 24 hours after treatment.
The overall situation and the homology target thing of C.miRNA inhibitor disinthibite
Carry out the specificity that full-length genome microarray sequence type analysis makes target thing disinthibite to measure miR-15 inhibitor.Particularly, from the heart tissue of the mice through M-11214 process and the mice through saline treatment, whole RNA is extracted.RNA is subsequently through full-length genome microarray sequence type analysis.By through M-11214 process the whole RNA of heart with compared with whole RNA of saline treatment time the gene that raises in, (the p value: 0.01 of the enrichment in the Genomic Imprinting (signature) raised of the gene containing miR-15 seed; Figure 11).Result confirms, M-11214 causes the target gene of miR-15 family specificity to disinthibite.
realexecute
example 5: the activity in vivo of miR-15 inhibitor in rat
Test the activity in vivo of miR-15 inhibitor in rats further.More specifically, 25mg/kg miR-15 inhibitor is injected to S-D (Sprague-Dawley) rat (49-52 age in days), comprises M-10670, M-11211, M-11213, M-11214, M-11215, M-11220, M-11221, or M-11222.Only give single dose.Within 4th day, collect heart tissue after injection, and analyze expression of target gene.Result display (Figure 12 A), 16 aggressiveness inhibitor seem in rats to expression of target gene having more effective effect.
Result also shows that Pim1 is a new miR-15 family gene target thing.The process LAN previously having shown Pim1 causes the proliferation activity of cardiac precursors (CPC) to increase.CPC produces the self renewal cell of daughter cell, and these daughter cells to the new myocyte of heart supply and blood vessel, and then stimulate Myocardial Regeneration.Contacting between miR-15 and Pim1 shows, suppresses miR-15 to make to produce new myocardial cell, and it may be useful for the loss of reparation heart.
In addition, compare research with check miR-15 inhibitor in rats with the effect in mice.As previously noted, with miR-15 inhibitor treatment S-D rat, and the expression of miR-15 target gene in 48 hours analysis of cardiac tissues after injection.As shown in Figure 12B, except seeming in rats except invalid 12 aggressiveness inhibitor, miR-15 inhibitor seems in rats than having more effective effect in mice.
realexecute
the activity in vivo of example 6:miR-15 inhibitor in ischemia-reperfusion model
A.miR-15 inhibitor makes target gene disinthibite
The activity in vivo of miR-15 inhibitor is tested in ischemia-reperfusion model.Particularly, in ischemia/reperfusion model, to the miR-15 inhibitor injecting single dose (25mg/kg) in rat vein, comprise M-10670, M-11211, M-11214, or M-10591.Within 72 hours after Reperfu-sion, put to death rat, and collection organization and analysis expression of target gene (as measured by PCR in real time).As shown in Figure 13, when with non-infraction matched group (time compared with baseline), all miR-15 inhibitor are all induced and are comprised Birc5, Cdc2a, and significantly the disinthibiting of the target gene of Granulin.
B.miR-15 inhibitor suppresses Markers of inflammation
Ischemical reperfusion injury makes myocardial infarction marginal area comprise VCAM, ITGAM, and the mrna expression of the various Markers of inflammations of CD40 raises.The impact that miR-15 inhibitor is expressed Markers of inflammation in this marginal area is shown in Figure 14.
C.miR-15 inhibitor makes myocardial infarction reduce
This research have evaluated the impact of miR-15 inhibitor on myocardial infarction.Particularly, after ischemia-reperfusion 3 days, risk area and myocardial infarction size is analyzed.The impact of miR-15 inhibitor on risk area and myocardial infarction size is respectively illustrated in Figure 15 A and 15B.
D.miR-15 inhibitor makes ejection fraction improve
This research uses the impact of miR-15 inhibitor on ejection fraction of ischemia-reperfusion model evaluation.Particularly, when Reperfu-sion and after Reperfu-sion one week, two weeks and three weeks, to the miR-15 inhibitor injecting single dose (25mg/kg) in rat vein, M-11211 and M-11214 is comprised.As shown in Figure 16, in the rear surrounding of ischemical reperfusion injury, when with compared with the animal of saline treatment time, miR-15 inhibitor makes ejection fraction significantly improve.
Claims (45)
1. the oligonucleotide containing at least one lock nucleotide,
Wherein said oligonucleotide comprises nucleotide sequence 5 '-GTGCTGCT-3 ', and complementary in fact with the nucleotide sequence of one or more miR-15 family member, and
Wherein said oligonucleotide in vitro in luciferase assay method under the oligonucleotide concentration of about 50nM or less, or in rodent model under the dosage of about 25mg/kg or less, reduce or suppress the activity of one or more miR-15 family member.
2. oligonucleotide according to claim 1, wherein said one or more miR-15 family member is selected from miR-15a, miR-15b, miR-16, miR-195, miR-424 and miR-497.
3. the oligonucleotide described in claim 1 or 2, it contains at least 8 lock nucleotide.
4. the oligonucleotide of any one of claim 1-3, the length of wherein said oligonucleotide is about 8 to about 18 nucleotide.
5. oligonucleotide according to claim 4, the length of wherein said oligonucleotide is about 12 to about 17 nucleotide.
6. oligonucleotide according to claim 5, the length of wherein said oligonucleotide is about 16 nucleotide.
7. the oligonucleotide described in claim 5 or 6, it comprises and is selected from following nucleotide sequence: 5 '-ACCATTATGTGCTGCT-3 ' (SEQ ID NO.1), 5 '-ACCATGATGTGCTGCT-3 ' (SEQ ID NO.2), 5 '-ATATTTACGTGCTGCT-3 ' (SEQ ID NO.3) and 5 '-ATATTTCTGTGCTGCT-3 ' (SEQ ID NO.4).
8. the oligonucleotide of claim 6 or 7, it contains 9 lock nucleotide and/or 7 non-lock nucleotide.
9. the oligonucleotide of any one of claim 6-8, wherein at least position 1,5,8,10 and 16 is lock nucleotide.
10. the oligonucleotide of claim 4, the length of wherein said oligonucleotide is about 12 nucleotide.
The oligonucleotide of 11. claim 4 or 10, it comprises and is selected from following nucleotide sequence: 5 '-TTATGTGCTGCT-3 ' (SEQ ID NO.5), 5 '-TTACGTGCTGCT-3 ' (SEQ ID NO.6), 5 '-TTCTGTGCTGCT-3 ' (SEQ ID NO.7), 5 '-TTCCGTGCTGCT-3 ' (SEQ IDNO.8), 5 '-TGATGTGCTGCT-3 ' (SEQ ID NO.9), 5 '-TGACGTGCTGCT-3 ' (SEQ ID NO.10), 5 '-TGCTGTGCTGCT-3 ' (SEQ ID NO.11), with 5 '-TGCCGTGCTGCT-3 ' (SEQ ID NO.12).
The oligonucleotide of 12. claim 10 or 11, it contains 8 lock nucleotide and/or 4 non-lock nucleotide.
Oligonucleotide any one of 13. claim 10-12, wherein at least position 1,4,9 and 12 is lock nucleotide.
The oligonucleotide of 14. claim 4, the length of wherein said oligonucleotide is about 8 nucleotide.
The oligonucleotide of 15. claim 14, it has all in fact lock nucleotide and nucleotide sequence 5 '-GTGCTGCT-3 '.
The oligonucleotide of 16. any one of claim 1-15, wherein comprises at least 4 with the region of the seed zone complementation of described one or more miR-15 family member and locks nucleotide.
The oligonucleotide of 17. any one of claim 1-16, wherein said oligonucleotide is containing having non-lock nucleotide continuous in 3 and/or the continuous nucleotide section of locking nucleotide more than 3.
The oligonucleotide of 18. any one of claim 1-17, wherein at least one non-lock nucleotide is selected from 2 ' deoxy, 2 '-O-methyl, 2 '-O-alkyl, 2 '-halogen and 2 '-fluorine-based nucleotide.
The oligonucleotide of 19. claim 18, wherein at least one non-lock nucleotide is 2 ' deoxy.
The oligonucleotide of 20. claim 19, wherein all non-lock nucleotide is 2 ' deoxy.
Oligonucleotide any one of 21. claim 1 to 20, wherein this lock nucleotide has 2 ' to the 4 ' bridge comprising vinyl or methylene.
The oligonucleotide of 22. claim 21, wherein said lock nucleotide has 2 ' to 4 ' methylene bridge.
Oligonucleotide any one of 23. claim 1-22, it has 5 ' and/or 3 ' cap.
Oligonucleotide any one of 24. claim 1-23, it contains one or more thiophosphate and connects.
The oligonucleotide of 25. claim 24, wherein said oligonucleotide is that complete thiophosphate connects.
The oligonucleotide of 26. claim 24, it has 1 to 3 phosphate ester and connects.
The oligonucleotide of 27. claim 1, it has the structure of following compound: M-10113, M-10134, M-10564, M-10566, M-10567, M-10670, M-11206, M-11207, M-11208, M-11209, M-11210, M-11211, M-11212, M-11213, M-11214, M-11215, M-11216, M-11217, M-11218, M-11219, M-11220, M-11221, M-11222, M-11223, or M-11224.
The oligonucleotide of 28. any one of claim 1-27, it comprises the lipophilic group of side joint further.
29. 1 kinds of pharmaceutical compositions, it includes the oligonucleotide of any one of claim 1-28 of effective amount, or its pharmaceutically acceptable salt, and pharmaceutically acceptable carrier or diluent.
The pharmaceutical composition of 30. claim 29, wherein said pharmaceutically acceptable carrier comprises dispersion system of colloid, macromolecular complex, Nano capsule, microsphere, beadlet, oil in water emulsion, micelle, mixed micelle or liposome.
The pharmaceutical composition of 31. claim 29, wherein said pharmaceutically acceptable carrier or diluent are made up of saline substantially.
32. 1 kinds are reduced or the method for activity of one or more miR-15 family member in T suppression cell, comprise and described cell is contacted with the oligonucleotide any one of claim 1-28 or the compositions any one of claim 29-31.
The method of 33. claim 32, wherein said cell is mammalian cell.
The method of 34. claim 33, wherein said cell is heart cell.
The method of 35. claim 32, wherein said cell in vivo or in vitro.
36. prevent or treat a method for the situation that is relevant to one or more miR-15 family member or that mediated by one or more miR-15 family member in experimenter, the dosage comprising to be enough to make the target thing of Pim1 to disinthibite uses at least one miR-15A family group inhibitor to this experimenter.
The method of 37. claim 36, wherein said miR15 family group inhibitor is the oligonucleotide described in any one of claim 1-28.
The method of 38. claim 36, comprises and uses to this individuality the pharmaceutical composition that right wants described in any one of 29-31.
The method of 39. claim 38, wherein uses described pharmaceutical composition by parenteral administration or by being injected directly in heart tissue.
The method of 40. claim 39, wherein said parenteral administration is intravenous, subcutaneous, intraperitoneal or intramuscular.
Method any one of 41. claim 38-40, wherein by oral, percutaneous, sustained release, Co ntrolled release, delayed release, suppository, conduit or sublingual administration applying said compositions.
Method described in 42. any one of claim 36-41, wherein said situation is heart.
The method of 43. claim 42, wherein said heart is pathological heart hypertrophy, myocardial infarction, heart failure, ischemia or ischemic damage and reperfusion damage.
The method of 44. claim 42, wherein said heart is relevant to heart transplantation.
The method of 45. any one of claim 36-44, wherein said experimenter is the mankind.
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KR20150036140A (en) | 2015-04-07 |
EA201590070A1 (en) | 2015-04-30 |
US20130345288A1 (en) | 2013-12-26 |
HK1209621A1 (en) | 2016-04-08 |
SG11201408460UA (en) | 2015-01-29 |
AU2013277033A1 (en) | 2015-01-22 |
TW201406774A (en) | 2014-02-16 |
JP2015525081A (en) | 2015-09-03 |
EP2863956A1 (en) | 2015-04-29 |
IN2014DN10899A (en) | 2015-09-11 |
BR112014032239A2 (en) | 2017-08-01 |
MX2014015641A (en) | 2015-07-14 |
US9163235B2 (en) | 2015-10-20 |
EP2863956A4 (en) | 2016-01-20 |
WO2013192486A1 (en) | 2013-12-27 |
AR091539A1 (en) | 2015-02-11 |
MD20150006A2 (en) | 2015-06-30 |
CA2876105A1 (en) | 2013-12-27 |
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